Thesis

Influenza is an acute respiratory disease caused by infection with influenza virus and is a major public health
problem. A better understanding of the interaction between influenza virus and host allow us to better understand the pathophysiology of influenza infection, and thus, ultimately, to better
protect themselves against the disease. Morbidity and mortality caused by severe influenza infections are associated with dysregulation of the immune response in the lung. This deleterious
inflammation is the cause of lung collateral damage, causing a decrease in the patient's breathing capacity. Although the mechanisms involved are not fully understood, recent studies point to a
central role of endothelial cells in the deregulation of the host response to influenza infection. During endothelium aggression, the physiological process of hemostasis (platelet activation,
coagulation and fibrinolysis) is activated in order to allow wound healing and to maintain the integrity of blood vessels. In many inflammatory diseases, the only dysregulation of hemostasis is
directly linked to a deleterious inflammatory response. During my thesis, we hypothesized that hemostasis could be the cause of the inflammatory dysregulation during influenza infections. Our
data show the role of two factors strongly involved in hemostasis: the thrombin activated receptor, PAR-1 (protease activated receptor 1) and plasminogen, in the deleterious lung inflammation and
in the pathogenicity of influenza virus. Besides the role of hemostasis, we have also been able to show that the influenza virus incorporates cellular proteins in the viral envelope, allowing it
to evade the immune system, which could also contribute to the deregulation of the host response. All the results obtained allowed to better understand the mechanisms involved in immune response
dysregulation during influenza infection and suggest new therapeutic targets to fight against the disease.

[Étude de la pathogénicité des virus Influenza A/H1N1].

Jean-Sébastien CASALEGNO - March 2014.

Influenza is responsible of 2,5 million cased of respiratory infection, each winter, in France. It is a main
human health threat and a main public health concern. The current treatment of flu relies on antiviral drug targeting viral proteins that can induce the appearance of resistant virus. Until 2007,
less than 5% of all circulating Influenza strains were resistant to neuraminidase inhibitors. Against all odds, Oseltamivir resistant A/Brisbane/59/2007(H1N1) emerged and spread during 2007/2008
and 2008/2009 season. To understand the driving force of this emergence we tested clinical strains from 2007 to 2011 for Oseltamivir and Zanamivir resistance, sequenced their HA and NA gene, and
compared their NA enzymatic properties with A(H1N1) reference strain from 1977 to 2007. Our results showed that A/Brisbane/59/2007 displayed a high affinity compare with NA of previous A(H1N1)
reference strains. Moreover these results suggested that HA-NA balance underlie the worldwide emergence of the Oseltamivir resistance without any selection pressure. The A(H1N1)pdm09 pandemic
highlighted the importance of lower tract respiratory infection in the severe influenza case. The high proportion of viral pneumonia observed during the 2009 pandemic and the following season
suggest that A(H1N1)pdm harbored a virulence factor that allowed the virus replication in the lower respiratory tracts. We studied the effect of HA 222 polymorphism on HA properties, HA-NA
balance and in vitro and in vivo Influenza phenotype. Our results demonstrated that G222, E222 or N222 HA mutation increased the binding of A(H1N1)pdm09 mutation to the lower respiratory tract
receptor. This may explain the A(H1N1)pdm09 increase pathogenicity. Moreover our results show than an optimal HA-NA balance is link to an efficient replication profile in
vitro.

Human rhinoviruses (HRV) remain a significant public health problem as they are the major cause of both upper and
lower respiratory tract infections. To date no vaccine or antiviral are available against these pathogens. Using a high-throughput yeast two-hybrid screening, we identified a six amino acid “hit”
peptide, LVLQTM, which acted as a pseudo-substrate of the viral 2A cysteine protease (2Apro) and inhibited its activity. This peptide was chemically modified at its C-terminus with a reactive
electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a
strong inhibitor of HRV replication in both A549 cells and mice. Based on HRV-2 2Apro crystallographic data, a virtual docking model was then set up to predict the inhibitor binding mode into the
ligand binding pocket of the enzyme. Sequence comparison between different 2Apro from HRV-A, -B and –C species revealed that the aminoacid residues involved in the interaction with the inhibitor
are relatively well conserved. If our peptide inhibitor seemed to be of general use against all HRV serotypes, its use for therapeutic purposes could be extended to other enterovirus-associated
diseases since it was also active against Human Enterovirus 71 (EV-71) 2A proteases and EV-71 replication. Moreover, comparison of the sequence of these proteases with the one of HRV-A2 revealed
only minor differences in the residues involved in the interaction with LVLQTM. Therefore, this study opens new doors in the development of an antiviral against a wide range of
enteroviruses.

The annual seasonal flu caused by influenza viruses can affect 5 to 15 % of the population. In addition to
vaccination, the use of antiviral drugs in the treatment and prevention allows the control of influenza virus infection. So far, two classes of antiviral drugs have been approved for influenza
treatment, one to inhibit the uncoating step (M2 inhibitors), and the other to prevent the release of progeny virions (NA inhibitors). However, the emergence and circulation of M2 inhibitor
resistant viruses at high frequencies have restricted the use of these inhibitors. Neuraminidase inhibitor resistance among circulating influenza viruses has emerged since the 2008 – 2009 season.
The development of new classes of antiviral agents is crucial in the fight against influenza virus. In recent years, many molecules belonging to a large group of compounds known as carbohydrates
(oligosaccharides/polysaccharides) have been revealed essential for various biological activities. The establishment of carbohydrate-based antiviral agents is, therefore, a highly promising
strategy. In order to evaluate the potentially anti-influenza molecules derived from carbohydrates, we have performed a screening from a library of 245 polysaccharides and oligosaccharides. These
compounds were extracted mainly from plants and algae. Several active molecules of different families have been identified. Among them, the sulphated oligosaccharide 152, belonging to the family
of arabinogalactane, was found to be highly active toward both influenza virus A and B in vitro. This oligosaccharide was purified from the green algal species Codium fragile. The study of the
152 mechanism suggests that this oligosaccharide can cooperatively inhibit both viral HA binding activity and NA catalytic activity.

Each winter, influenza epidemics have a considerable impact on the population in terms of morbidity and mortality.
Influenza A virus is the main etiologic agents of influenza. They present at their surface two glycoproteins, the neuraminidase and the hemagglutinin. These two proteins have antagonist functions
: the hemagglutinin allows the virus to enter the host cell and the neuraminidase, through its sialidase activity, releases progeny virions from host cells. Although prophylaxis of influenza is
mainly based on vaccination, antiviral drugs play a very important role in the fight against epidemics of influenza and the strategy developed in anticipation of a flu pandemic. The neuraminidase
inhibitors are effective antiviral against influenza. Through the inhibition of the neuraminidase enzymatic activity, they prevent the release of new virions formed. The introduction into
clinical practice of new drugs requires monitoring in order to detect the potential emergence of resistance. Although the approach to the design of neuraminidase inhibitors has provided hope that
resistance will be limited, resistance to NAIs already been observed in clinical, encouraging close monitoring. It is therefore important to continue to study and understand the various
mechanisms of resistance to neuraminidase inhibitors. The work of this thesis has thus focused on understanding the diversity of resistance mechanisms. Initially, we studied the impact of
mutations in all structural residues of the active site of neuraminidase. We observed that most of these mutations did not alter the characteristics of the virus and induced very limited
resistance to antivirals. Subsequently, we then sought to explore opportunities for synergy in resistance by the combination of two structural mutations of the active site of neuraminidase. On
four viruses produced, only the virus with the double mutation E119V+I222L in the active site of neuraminidase was viable, although its in vitro replicative capacity was impaired. The combination
of these two mutations induced a synergistic resistance to oseltamivir. Finally, we wanted to integrate the functional interaction of neuraminidase with hemagglutinin. We have shown that the
combination of a hemagglutinin low affinity for sialylated receptors allowed to rescue a virus with a deficient neuraminidase. Thus an influenza virus may discharge the function of neuraminidase,
the target of the only available effective antivirals. The mechanisms of resistance to neuraminidase inhibitors are numerous. Plus, the circulation in the last two seasons of resistant viruses
without selective pressure challenges the assumptions developed on the possible emergence of resistance in clinic. This opens new issues to consider in order to understand the mechanisms that
allowed this emergence and transmission.

In the context of A(H5N1) pandemics threat, an « avian flu and flu pandemics » project was proposed by LyonBioPole
to develop influenza viruses characterization tools for vaccine production. To study genetic reassortment between influenza viruses, 3 reverse genetic systems of A(H3N2) human virus and A(H5N2)
and A(H5N1) avian viruses were developed and reassortant viruses were produced. Their replicative capacities were evaluated using growth kinetics on MDCK cells with viral production
quantification by real-time qRT-PCR. The A(H1N1)2009 emergence raises two questions about the acquisition by genetic reassortment of oseltamivir resistance and/or pathogenicity determinants. A
co-infection protocol on MDCK cells was developed to study gene constellations of reassortant viruses like A(H1N1)2009-A(H1N1) H275Y and A(H1N1)2009-A(H5N1). We report here that genetic
reassortment is possible between avian, human and swine strains using reverse genetic and viral co-infection and that some specific constellations emerged. We also report, that pandemic
A(H1N1)2009 can acquire the H275Y mutated NA from seasonal oseltamivir resistant A(H1N1) viruses without any modifications on replicative capacities. This genetic reassortment is also possible
with A(H5N1) viruses. These observations strenght the importance of vaccination against all these influenza strains to reduce the risk of one-individual viral co-infection.